GBA activity (‘BGL test’)

Hypothesis: Intracellular trafficking of GBA, MOA of silent chaperone ^{(Takeda confidential)}

1. In ER, GBA forms a dimer and binds to LIMP-2 dimer. Cell 131, 770. and PNAS 113, 3791.
2. After trafficking into lysosome, LIMP-2 dissociates the GBA dimer under the acidity. Cell 131, 770. and PNAS 113, 3791.
3. GBA forms a monomer by the interaction of Sap C existing only in lysosome. Biochemical and Biophysical Research Communications 457, 561.
4. Monomeric GBA shows enzymatic activity. (The activity is enhanced by Sap C.)

• (2018 Medscape) glucocerebrosidase activity in peripheral blood leukocytes. a standard blood test called a β-glucosidase leukocyte (BGL) test
• Most major labs, such as Counsyl, Labcorp and Quest Diagnostics, are familiar with a β-glucosidase leukocyte (BGL) enzyme assay and have historically been able to perform the test.
• A finding of less than 15% of mean normal activity or < 8.7 nmol/h/mg protein (https://gaucherdiseasenews.com/bgl-test/), or 4.6 - 12.0 nmol hydrolyzed/hr/mg protein (ARUP) is diagnostic of GD.
• Heterozygotes generally have half-normal enzyme activity, but as much as 20% overlap with activity levels of healthy controls has been reported (Essabar, 2015 #847),
• Substrate
  • production of fluorescent 4-methylumbelliferone (4-MU) from 4-methylumbelliferyl-b-D-glucopyranoside (4-MUG = 4-MUBG) substrate.
• Normal range

	CSF	CSF	DBS	DBS	DBS	DBS
	내 뒤 총정리에 있는 것 중 하나 아닐까?	Prevail Therapeutics Reports Second Quarter 2020 Financial Results (20200811)	Kang 2017	(Kang, 2017 #854)	(Alcalay, 2015 #258)	PPMI 2019 investigator presentation
control			New born	116 healthy individuals (2-46 years old)	N=252, mean age 65 y
method				Fluorogenic, 4MU, Inhouse method	Synthetic substrate(not disclosed), internal method, MS	Sanofi GCaseassay validated for dried blood spot (DBS) PPMI stores frozen WB; WB from 66 subjects spotted fresh and after freeze-thaw »Paired samples analyzed side by side in SANOFI GBA MS/MS assay
GBA activity in the control		(adult normal range: 1.1 - 8.1 μmol/L/d).	35 to 140 nmol/h.ml Min 30.47, mean 145.69, max 479.52, sd 44.76	Mean 59.38, s.d.19.69, Min 30.59, Max 153.48, Median 56.75, percentile 25: 46.67, percentile 75: 67.16	mean 11.85, SD 3.40 (so 8.45 - 15정도)	scatter plot `GBA activity in DBS (uM/h)` vs `GBA Activity in FB DBS (uM/h)` alcalay와 비슷하네. (노인이라 그럴듯)
Overlap with heteroz?				(heteroz n=19) No if we compare using quartile 2&3 for both. , No if we compare using mean±one SD (40-80 vs 20-40) Yes if we compare using 95% prediction interval (mean+/- 2SD): 10-50 vs 20-100 (this is probabaly used for author's conclusion) Author's conclusion: we found an overlap in the GBA activity reference range between normal newborns and obligate carriers of GBA gene mutations.	노인에서는 GBA activity value 가 훨씬 적구나!

DBS: has not replaced conventional whole blood, plasma, or serum samples.

Sebastian 20201124

		Sample	Substrate	Method	Notes / results		Presented unit
Ortega et al	2016	Fresh blood	4-MUBG	Unclear			~5-55 nmol/mg protein / hr
ashrazm et al	2018	PBMCs	PFB-FDglu	Flow cytometry-based	Weak separation between PD and healthy donor in monocytes only		~5-40 Arbitrary unit
Wolf et al	2017	Dried blood spot	4-MUG	20h incubation, fluorescence	Gaucher's disease vs HD: 100% sensitivity, 98% specificity		~5 - 30 μmol/L/h
Wolf et al	2017	Dried blood spot	C12	20h incubation, mass spec	Gaucher's disease vs HD: 100% sensitivity, 100% specificity		~5 - 30 μmol/L/h
ollins et al	2017	Skin fibroblasts cell lysates	4-MUBG	1h incubation, fluorescence	Clear separation between GD and healthy donor		~50 - 250 nmol/h/mg
Lu et al	2010	Skin fibroblasts cell lysates	4-MUG	1h incubation, fluorescence	Clear separation between GD and healthy donor		~20 - 20 % of normal
nderson et al	2020	Cell / tissue lysate		1h incubation, fluorescence
rnetti et al	2014	CSF (50μL)	4-MUBG	24h incubation, fluorescence	Activity correlated to PD staging		~0.1 – 1.7 mU / mL
sichetti et al	2014	CSF (20μL)	4-MUBG	3h incubation, fluorescence	Methodological paper comparing storage conditions	sPD	~1.5 – 15 mU / L (눈대중일듯)
ullin et al	2020	CSF (20μL) x 10	4-MUBG	3h incubation, fluorescence	Ambroxol paper. Some reduction of activity after treatment	GBA-PD & GBA+PD combined	~0.1 – 0.8 nmol/mL/h (눈대중)
μmol/L/hr 과 같은단위네
iques et al	2017	Tissue lysate	b-NPG (commercial kit)	20 min incubation, measure	CBE treatment reduced activity		~100-220 U/μg protein
vail report	2020	CSF	?	?	Activity increased at 4 months after treatment	정상치라함	1.1 – 8.1 μmol/L/d (근거무)

Product (kit)

Company	Sigma-Aldrich	SEMA4	ANASPEC, SensoLyte® Red Glucocerebrosidase (GBA) Assay Kit This is for research, not for human!	LABCORP	ARUP
	https://www.sigmaaldrich.com/catalog/product/sigma/mak129?lang=ja®ion=JP	https://sema4.com/products/test-catalog/acid-%CE%B2-glucosidase-gaucher-disease/	https://www.anaspec.com/products/product.asp?id=59067	https://www.labcorp.com/tests/451780/gaucher-disease-enzyme-analysis-glucocerebrosidase	https://ltd.aruplab.com/Tests/Pub/2014459
	colorimetric tests β-glucosidase activity, β-Gurukoshi Daze is p - nitrophenyl -β-D- glucopyranoside hydrolyzed, available colorimetric (4 05 nm), anti-producing product that is proportional to the existing β-glucosidase activity is measured by the response. One unit of β-glucosidase is the amount of enzyme that catalyzes the hydrolysis of the substrate at pH 7.0 at 1.0 μmole per minute. Sebastian started from this	measured with a lysate from the patient's (WBC) by a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method. Specimen requirements: Anticoagulated blood: 5-10 mL in sodium heparin tube (green top) or 1-2 leukocyte pellets	Fluorogenic substrate. The GBA substrate provided in the kit releases the red fluorescent dye resorufin upon Glucocerebrosidase cleavage with absorption/emission maxima at 570 nm/610 nm.	fluorometric enzyme assay using 4-methylumbelliferyl-β-D-glucosida as substrate, whole blood is used	Quantitative Fluorometry, whole blood
Turnaround time		7 days		7-13 dyas
These are 'research only' so not clinically validated, or FDA-approved? Not companion-diagnositc (근데 위기술보면, whole blood에서, 진단치도 있고, normal치도 있는것 같은데?)

• Patient selection: As whether GBA activity is actually reduced is not established in some GBA variants, we may confirm GBA deficiency in participants if we measure GBA activity in individuals and pre-define cut points.

• Additionally, Patient enrichment strategy The hypothesis that GBA-PD patients with significantly reduced GBA activity (<x %) may be validated clinically. I envisioned a clinical scenario below for GBA-targeting treatments. A question would be: would we need any preclinical support to work on the hypothesis in addition to our planned preclinical POC plan? GBA-PD trial, upon entry to the trial, categorizing the participants according to GBA activity level on predefined cut-points, and subsequent enrichment of subpopulation based on differentiated treatment response-if any.

Uncertain Spans

location	transcription	uncertainty
Normal range / Kang Mean 59.38...percentile 75: 67.16	red text run	small-font dense numbers; values are read directly from the visible cell.
Sebastian table / 1st column author names	ashrazm, ollins, nderson, rnetti, sichetti, ullin, iques, vail report	the leftmost author column is partially clipped by the photo edge; first letter(s) are not visible.
Patient selection paragraph	right-edge clipping	the red Patient-selection / Patient-enrichment paragraphs run off the right edge of this capture.